过表达miR-124a对J774.1细胞中TNF-α和IL-6表达水平及细胞周期的影响

doi:10.13481/j.1671-587x.20180517

吉林大学学报(医学版) ›› 2018, Vol. 44 ›› Issue (05): 983-987.doi: 10.13481/j.1671-587x.20180517

过表达miR-124a对J774.1细胞中TNF-α和IL-6表达水平及细胞周期的影响

尹芳蕊, 庞春艳, 耿立霞, 王永福

包头医学院第一附属医院风湿免疫科包头医学院风湿免疫研究所内蒙古自治区自体免疫学重点实验室, 内蒙古包头 014010

收稿日期:2018-01-06 出版日期:2018-09-28 发布日期:2018-11-20
通讯作者: 王永福,主任医师,硕士研究生导师(Tel:0472-2178469,E-mail:yfrjiayou@yeah.net) E-mail:yfrjiayou@yeah.net
作者简介:尹芳蕊(1981-),女,内蒙古自治区包头市人,主管检验师,医学硕士,主要从事风湿免疫疾病实验室检测方面的研究。
基金资助:
内蒙古自治区卫计委医疗卫生科研计划项目资助课题（201301070）

Effects of miR-124a overexpression on expression levels of TNF-α and IL-6 and cell cycle of J774.1 cells

YIN Fangrui, PANG Chunyan, GENG Lixia, WANG Yongfu

Department of Rheumatology, First Affiliated Hospital, Baotou Medical College;Institute of Rheumatology and Immunology, Baotou Medical College;Inner Mongolia Key Laboratory of Autoimmunity;Baotou 014010, China

Received:2018-01-06 Online:2018-09-28 Published:2018-11-20

摘要/Abstract

摘要： 目的：探讨miR-124a对类风湿关节炎（RA）细胞模型小鼠巨噬细胞J774.1细胞中肿瘤坏死因子α（TNF-α）和白细胞介素6（IL-6）表达水平及细胞周期的影响，阐明miR-124a在RA中的作用机制。方法：取对数生长期J774.1细胞，以1×10⁶mL^-1均匀接种于培养皿，每组设3个复孔，当细胞融合度达到60%时进行感染，实验分为miR-124a过表达组（感染miR-124a腺病毒载体）、空载腺病毒组（感染阴性病毒液）和空白对照组（不做任何处理）。ELISA法检测感染48 h后3组细胞上清液中TNF-α和IL-6的表达水平，RT-PCR法检测感染48 h后J774.1细胞中TNF-α和IL-6 mRNA相对表达水平，流式细胞术检测感染48 h后3组细胞细胞周期变化。结果：感染48 h后，与空白对照组和空载腺病毒组比较，miR-124a过表达组细胞上清中TNF-α和IL-6表达水平明显降低（P=0.038，P=0.042；P=0.043，P=0.044），miR-124a过表达组细胞中TNF-α和IL-6 mRNA表达水平明显降低（P=0.001，P=0.002；P=0.001，P=0.003），G₁期细胞百分率明显升高（P=0.01），S期和G₂期细胞百分率明显降低（P<0.01）。结论：miR-124a感染小鼠巨噬细胞J774.1后可使细胞的炎性因子表达水平下降，抑制细胞增殖，miR-124a有望成为RA治疗的新靶点。

关键词: miR-124a, 类风湿性关节炎, 腺病毒载体, J774.1细胞

Abstract: Objective:To explore the effects of miR-124a on the expression levels of tumor necrosis factor alpha (TNF-α)and interleukin-6(IL-6) and cell cycle in the macrophage J774.1 cells of the rheumatoid arthritis(RA) model mice, and to elucidate the mechanism of miR-124a in the pathogenesis of RA. Methods:The J774.1 cells in logarithmic growth phage were obtained and uniformly inoculated in the petri dish with 1×10⁶ mL^-1,and there were 3 multiple holes in each group;transfection was carried out when the fusion degree of the cells reached 60%.The cells were divided into blank control group(without any treatment), empty vector group(transfected with adenovirus negative fluid)and miR-124a overexpression group(transfected with miR-124a adenovirus vector).The expression levels of TNF-α and IL-6 in supernatant of the cells in three groups were detected by ELISA 48 h after transfection. RT-PCR was used to detect the relative expression levels of TNF-α and IL-6 mRNA in J774.1 cells 48 h after transfection and the changes of cell cycle were detected using flow cytometry. Results:The expression levels of TNF-α and IL-6 in supernatant of the cells in miR-124a overexpression group were lower than those in blank control group and empty vector group 48 h after transfection(P=0.038,P=0.042;P=0.043,P=0.044).The expression levels of TNF-α mRNA and IL-6 mRNA in miR-124a group were significantly lower than those in blank control group and empty vector group(P=0.001,P=0.002;P=0.001,P=0.003).The pecentage of cells in G₁ phase in miR-124a overexpression group was higher than those in blank control group and empty vector group(P<0.01);the percentages of cells in S phase and G₂ phase were lower than those in blank control group and empty vector group(P<0.01). Conclusion:MiR-124a can decrease the expression levels of inflammatory cytokines after transfecting the macrophages J774.1 cells,and inhibit the proliferation of cells.MiR-124a is a potential therapeutic target for the treatment of RA.

Key words: microRNA-124a, rheumatoid arthritis, adenovirus vector, J774.1 cells

中图分类号:

R329.28

尹芳蕊, 庞春艳, 耿立霞, 王永福. 过表达miR-124a对J774.1细胞中TNF-α和IL-6表达水平及细胞周期的影响[J]. 吉林大学学报(医学版), 2018, 44(05): 983-987.

YIN Fangrui, PANG Chunyan, GENG Lixia, WANG Yongfu. Effects of miR-124a overexpression on expression levels of TNF-α and IL-6 and cell cycle of J774.1 cells[J]. Journal of Jilin University(Medicine Edition), 2018, 44(05): 983-987.

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过表达miR-124a对J774.1细胞中TNF-α和IL-6表达水平及细胞周期的影响

Effects of miR-124a overexpression on expression levels of TNF-α and IL-6 and cell cycle of J774.1 cells

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